Modified microbubble

ABSTRACT

A modified microbubble is provided. The modified microbubble includes an albumin microbubble and a plurality of chitosan oligosaccharide lactates. The albumin microbubble includes an albumin shell and a gas core inside the albumin shell. The plurality of chitosan oligosaccharide lactates is connected to an outer surface of the albumin shell.

RELATED APPLICATIONS

The present application is a Divisional Application of the applicationSer. No. 15/093,757, filed Apr. 8, 2016, which claims priority toTaiwanese application serial number 105100346, filed Jan. 7, 2016, allof which are herein incorporated by reference.

BACKGROUND Field of Disclosure

The present disclosure relates to a modified microbubble.

Description of Related Art

Hair loss disorders generally affect men and women of all ages, and theimpact increases with age. Among disorders, androgenetic alopecia (AGA)is the most common form of hair loss, which arises from hair folliclesgenetically susceptible to androgens. In the susceptible hair follicles,dihydrotestosterone (DHT) binds to the androgen receptor, and thehormone-receptor complex then activates specific genes, which transformslarge, terminal follicles to small, miniaturized follicles.

To treat hair loss disorders, Minoxidil (Mx, with the product name ofRogaine®) is the only medication approved by the U.S. Food and DrugAdministration (FDA) that can be applied to both men and women so as toreduce hair loss and promote hair growth. However, constant use ofMinoxidil is required to work over the long term. Moreover, somepatients present with complaints of pruritus and inflammation of thescalp, and the major factor lies in the solvent, propylene glycol,instead of Minoxidil. Though many other solvent candidates areconsidered, such as butylene glycol, polysorbate, or glycerol, there isno guarantee that these organic solvents do not cause side effects.

On account of this, there is a need for a new prescription of Minoxidilthat enables the slowly release of Minoxidil in the target area and thetransdermal delivery of Minoxidil to the follicles, shortening thecourse of hair growth. Meanwhile, the new prescription does not adoptorganic solvents to reduce the immunogenicity and inflammations.

SUMMARY

The present disclosure provides a modified microbubble. The modifiedmicrobubble includes an albumin microbubble and a plurality of chitosanoligosaccharide lactates. The albumin microbubble includes an albuminshell and a gas core inside the albumin shell. The plurality of chitosanoligosaccharide lactates is connected to an outer surface of the albuminshell.

According to an embodiment of the present disclosure, the modifiedmicrobubble further includes a drug connected to the chitosanoligosaccharide lactate to form a drug-carrying modified microbubble.

According to an embodiment of the present disclosure, the drug has anegative charge.

According to an embodiment of the present disclosure, the drug isMinoxidil. The present disclosure provides a modified microbubble, whichcan be connected to drugs like Minoxidil, and releases drug throughultrasound treatment, enabling the transdermal penetration andabsorption by target tissues of drugs to shorten the course oftreatments.

According to an embodiment of the present disclosure, the modifiedmicrobubble has a diameter of 4,000 nm to 4,400 nm.

The present disclosure also provides a method of making a modifiedmicrobubble. The method includes providing an albumin microbubble andconnecting a plurality of chitosan oligosaccharide lactates to thealbumin microbubble in an environment with a temperature of 0-10° C. toform the modified microbubble.

According to an embodiment of the present disclosure, connecting theplurality of chitosan oligosaccharide lactates to the albuminmicrobubble in the environment with the temperature of 0-10° C. includesadding a first solution containing the plurality of chitosanoligosaccharide lactates to the albumin microbubble, and the pluralityof chitosan oligosaccharide lactates has a concentration of 1-5 mg/ml inthe first solution.

According to an embodiment of the present disclosure, the method furtherincludes connecting Minoxidil to the modified microbubble in anenvironment with a temperature of 0-10° C. to form a drug-carryingmodified microbubble.

According to an embodiment of the present disclosure, connecting theMinoxidil to the modified microbubble in the environment with thetemperature of 0-10° C. comprises adding a second solution containingMinoxidil to the modified microbubble to form a third solutioncontaining the drug-carrying modified microbubble, and the Minoxidil hasa concentration of 1-5 mg/ml in the second solution, and a volume ratioof the first solution to the second solution is 1:1 to 3:1, and theMinoxidil of the drug-carrying modified microbubble has a concentrationof 0.1-0.5 mg/ml in the third solution.

According to an embodiment of the present disclosure, the method furtherincludes adjusting a pH of the third solution to 4-6.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure can be more fully understood by reading thefollowing detailed description of the embodiment, with reference made tothe accompanying drawings as follows:

FIG. 1 illustrates a cross-sectional view of a modified microbubble inaccordance with some embodiments of the present disclosure.

FIG. 2 illustrates a cross-sectional view of a drug-carrying modifiedmicrobubble in accordance with some embodiments of the presentdisclosure.

FIG. 3 illustrates a flow chart of making a drug-carrying modifiedmicrobubble in accordance with some embodiments of the presentdisclosure.

FIG. 4A illustrates a diagram of zeta potentials in accordance withExperimental examples 1-6 and Comparative example 1 of the presentdisclosure.

FIG. 4B illustrates a diagram of particle diameters against quantity inaccordance with Experimental examples 1 and 4 and Comparative example 1of the present disclosure.

FIG. 4C illustrates scanning electron microscopy images in accordancewith Experimental examples 1 and 4 and Comparative example 1 of thepresent disclosure.

FIG. 5 illustrates a diagram of absorbance spectra in accordance withExperimental examples 1′ and 4′ and Comparative examples 1′, 3, 4 and 5of the present disclosure.

FIG. 6 illustrates a diagram of cumulative release rates of Minoxidilagainst time in accordance with Experimental examples 7-10 of thepresent disclosure.

FIG. 7 illustrates scanning electron microscopy images of transdermaldelivery of fluorescent agents in accordance with Experimental example11 and Comparative examples 6 and 7 of the present disclosure.

FIG. 8 illustrates a diagram of cumulative penetration amounts ofMinoxidil against time in accordance with Experimental examples 4 and 12and Comparative examples 1, 4, 8 and 9 of the present disclosure.

FIG. 9 illustrates top-view observation images of hair growth of micebacks against time in accordance with Experimental example 12 andComparative examples 4, 8, 9 and 10 of the present disclosure.

FIG. 10 illustrates a diagram of hair growth rates of mice backs againsttime in accordance with Experimental example 12 and Comparative examples4, 8, 9 and 10 of the present disclosure.

FIG. 11 illustrates scanning electron microscopy histological images ofmice backs in accordance with Experimental example 12 and Comparativeexamples 4, 8, 9 and 10 of the present disclosure.

DETAILED DESCRIPTION

To comprehensively illustrate the content of the present disclosure indetails, references will now be made to embodiments of the presentdisclosure, examples of which are illustrated in the accompanyingdrawings. These are, of course, merely examples and are not intended tobe limiting. The examples or embodiments can be combined or substitutedunder preferable circumstances, and one example or embodiment can beaffiliated to other examples or embodiments without further illustrationor explanation. In the following descriptions, many specific details areelaborated for readers to fully comprehend the following embodiments.Nonetheless, the present invention can also be realized under theconditions without the specific details. In addition, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise.

The present disclosure provides a modified microbubble, which can beconnected to drugs such as Minoxidil, and act as an ultrasound contrastagent, slowly releasing Minoxidil through ultrasound sonication. Thisenables the constant release of Minoxidil by ultrasound sonication onceit reaches target tissues, and enables the transdermal delivery ofMinoxidil to follicles, achieving the effect of shortening hair growthtreatments and reducing immunogenicity and inflammations.

Referring to FIG. 1, it illustrates a cross-sectional view of a modifiedmicrobubble in accordance with some embodiments of the presentdisclosure. As shown by FIG. 1, the modified microbubble 10 includes analbumin microbubble (MB) 100 and a plurality of chitosan oligosaccharidelactates (COLs) 200. The albumin microbubble 100 includes an albuminshell 110 and a gas core 120 inside the albumin shell 110. In someembodiments, the plurality of chitosan oligosaccharide lactates (COLs)200 is connected to an outer surface of the albumin shell 110. In someembodiments, the gas core 120 includes a barely soluble gas, such asperfluoropropane (C₃F₈) or sulfur hexafluoride (SF₆).

Since the albumin carries negative charges, the zeta potential of thealbumin shell 110 is below zero, and can thus attract molecules withpositive charges. Chitosan oligosaccharide lactates (COLs) 200 containsmany amino groups (—NH₂), which adhere to protons to form —NH₃ ⁺ groupsin acidic solutions, making the COL 200 positively charged. Accordingly,the negatively charged albumin shell 110 can be connected to the COL 200through the electrical attraction, facilitating the COL 200 to bedistributed across the outer surface of the albumin shell 110, formingthe modified microbubble (COL-MB) 10, which is modified by COL 200.

Referring next to FIG. 2, it illustrates a cross-sectional view of adrug-carrying modified microbubble in accordance with some embodimentsof the present disclosure. In some embodiments, the positively chargedCOL 200 can be further connected to a drug 300 with negative charges. Insome embodiments, Minoxidil (Mx) contains a negatively charged oxygenatom, and is thus inclined to be attracted by the —NH₃ ⁺ groups of theCOL 200 and attached to the COL 200. Thus, as shown in FIG. 2, COLs 200on the COL-MB 10 can be further connected to the drug 300 (such as Mx),forming the drug-carrying modified microbubble (Mx-COL-MB) 20. It isnoted that since both the MB 100 and the Mx 300 carry negativelycharges, MB 100 cannot be directly attached to Mx 300, but ratherconnects to Mx 300 via the COL 200 with positive charges, which makesthe drug-carrying modified microbubble (Mx-COL-MB) 20 tend to beelectrically neutral.

Compared to conventional, injectable drug-carrying microbubbles wheredrug encapsulation is required to prevent drug decomposition after entryinto the blood, the drug-carrying modified microbubble (Mx-COL-MB) 20 ofthe present disclosure is externally applied, and thus the drug 300 doesnot need to be encapsulated, but just need to be attached to the outersurface of the modified microbubble 10. Upon contact with the skin andthe ultrasound stimulation, the drug 300 can be released for transdermalpenetration.

Referring to FIG. 3, it illustrates a flow chart of making adrug-carrying modified microbubble in accordance with some embodimentsof the present disclosure. In step 402, the method of making themodified microbubble 10 includes providing the albumin microbubble (MB)100. In various embodiments, 10-30% sterile human serum albumin (HSA)solution is diluted to 1-5% (w/v) HSA stock solution byphosphate-buffered saline (PBS). Then, the C₃F₈ or SF₆ gas flows into5-20 mL HSA stock solution and undergo a 1-5 min, 100-300 W ultrasoundsonication by a ultrasound sonicator, facilitating the albumin to formshells and encompass the C₃F₈ or SF₆ gas to form the albumin microbubble(MB) 100. After forming the MB 100, centrifugations are performed toremove the solution and albumins that do not form shells.

Subsequently, in step 404, a plurality of chitosan oligosaccharidelactates (COLs) 200 is connected to the MB 100 in an environment with atemperature of 0-10° C. to form the modified microbubble (COL-MB) 10. Insome embodiments, the COL 200 is dissolved in a first solution, and hasa concentration of 1-5 mg/ml in the first solution. The first solutioncan be saline or PBS. In some embodiments, step 404 means adding 1-5 mlof the first solution containing 1-5 mg/ml COL (with the molecularweight of 4,000-6,000) to the MB 100. Then, several times ofcentrifugations and washing are performed to wipe out the free COL 200,leaving the solution with only COL-MB 10.

Next, in step 406, Minoxidil (Mx) 300 is connected to the modifiedmicrobubble 10 in an environment with a temperature of 0-10° C. to forma drug-carrying modified microbubble (Mx-COL-MB) 20. In someembodiments, step 406 includes adding a second solution to the modifiedmicrobubble 10, to form a third solution. Mx 300 is dissolved in thesecond solution, and has a concentration of 1-5 mg/ml in the secondsolution. The second solution can be saline or PBS. In some embodiments,step 406 includes adding the second solution containing 1-5 mg/ml Mx(with the molecular weight of 209.25) in different volume ratiosrelative to the first solution, such as 1:1, 1:2, or 1:3, to thesolution containing COL-MB 10, and stirring the blended solution at therate of 10-200 rpm at 0-10° C. for 20-30 hours to form a third solutioncontaining the drug-carrying modified microbubble (Mx-COL-MB) 20. Then,several times of centrifugations and washing are performed to wipe outthe free Mx 300. In some embodiments, the Minoxidil 300 attached to thedrug-carrying modified microbubble 20 has a concentration of 0.1-0.5mg/ml in the third solution.

In step 408, a pH of the third solution containing the Mx-COL-MB 20 isadjusted to 4-6. In various embodiments, the pH adjustment is achievedby titration of acids such as the hydrochloric acid or bases such assodium hydroxide into the third solution to respectively elevate orlower the pH. In some embodiments, the pH of the third solution isadjusted to pH 4.5-5.5, similar to the pH of the human scalp.

EXAMPLES

The following examples are meant to elaborate the specific embodimentsof the present disclosure in details, and to facilitate those skilled inthe art to implement the present disclosure. The following examples arenot meant to limit the present disclosure.

Experimental Examples 1-6 and Comparative Examples 1-2

Alterations in Zeta Potentials and Particle Diameters Before and Afterthe Modification and Drug Carriage of Microbubbles

Referring to FIG. 4A, it illustrates a diagram of zeta potentials inaccordance with Experimental examples 1-6 and Comparative example 1 ofthe present disclosure.

TABLE 1 Mx Zeta Particle attaching potential diameter efficiency (mV)(μm) (%) Comparative MBs −3.87 ± 2.78 1.55 ± 0.27 — example 1Comparative MBs + Mx −0.43 ± 1.01 1.46 ± 0.30  2.55 ± 0.15 example 2Experimental COL-MBs 20.23 ± 1.20 4.15 ± 0.17 — example 1 (1:1)Experimental COL-MBs 24.98 ± 1.10 4.36 ± 0.05 — example 2 (2:1)Experimental COL-MBs 25.23 ± 2.60 4.25 ± 0.82 — example 3 (3:1)Experimental Mx-COL-  0.41 ± 1.73 4.50 ± 0.10 14.87 ± 0.03 example 4 MBs(1:1) Experimental Mx-COL- 20.54 ± 1.02 4.69 ± 0.27 11.68 ± 0.01 example5 MBs (2:1) Experimental Mx-COL- 21.10 ± 1.97 4.30 ± 0.12 10.90 ± 0.01example 6 MBs (3:1)

As shown in FIG. 4A and the above Table 1, Comparative example 1represented unmodified microbubbles (MBs) with the zeta potential of−3.87±2.78 mV. Comparative example 2 (not shown in FIG. 4A) representedunmodified microbubbles and unattached Minoxidil (MBs+Mx) with the zetapotential slightly raised to −0.43±1.01 mV, while the Mx attachingefficiency was only 2.55±0.15%.

By blending a fixed amount of MBs with COL in different volume ratios,COL-MBs with different contents of COL were formed. In detail, themethod of making Experimental examples 1-3 included adding differentvolumes of the first solution containing 2 mg/ml COL to the fixed amountof MBs (with the volume ratio of COL:MB to be 1:1, 2:1, and 3:1), andperforming blending at 4° C. for 24 hours to form the COL-MBs(1:1)(Experimental example 1), the COL-MBs (2:1)(Experimental example2), and the COL-MBs (3:1)(Experimental example 3). The zeta potentialsof Experimental examples 1-3 were 20.23±1.20 mV, 24.98±1.10 mV, and25.23±2.60 mV respectively, which exhibited that the negatively chargedMB could be attached to a great amount of positively charged COLs,resulting in a great increase in the zeta potentials of COL-MBs.

Subsequently, the second solution containing Mx was added to thesolutions with COL-MBs (i.e. Experimental examples 1, 2, and 3) to formthe third solution containing Mx-COL-MBs (i.e. Experimental examples 4,5, and 6). In detail, the method of making Experimental examples 4-6included adding different volumes of the second solution containing 2mg/ml Mx to Experimental examples 1-3 respectively, and performingblending at 4° C. for 24 hours. The volume ratios of the second solutionto the first solution in Experimental example 4-6 were 1:1, 1:2, and 1:3respectively. As shown in FIG. 4A and the above Table 1, the zetapotential of Experimental example 4 was greatly reduced to 0.41±1.73 mV,while the zeta potentials of Experimental examples 5 and 6 only slightlydecreased to 20.54±1.02 mV and 21.10±1.97 mV respectively. Thisindicates that when the solution volume ratio of Mx:COL is 1:1 (i.e.Experimental example 4), the COL can be attached to maximal amount ofthe Mx, with the Mx attaching efficiency reaching 14.87±0.03%, causingthe concentration of attached Mx in the third solution to reach about0.3 mg/ml, and the zeta potential to have the maximal slump to reduce tonear zero and electrically neutral to be easily dissolve in the saline(such as PBS).

Referring to FIG. 4B, it illustrates a diagram of particle diametersagainst quantity in accordance with Experimental examples 1 and 4, andComparative example 1 of the present disclosure. As shown in FIG. 4B andthe above Table 1, the particle diameter of Comparative example 1 wasabout 1550±270 nm, the particle diameter of Experimental example 1 wasin the range between 4,000 nm to 4,400 nm, or about 4150±170 nm,indicating great increase in particle diameter after the connection ofthe MB to the COL. The particle diameter of Experimental example 4 isabout 4500±100 nm, indicating no great increase in particle diameterafter the connection of the Mx to the COL.

Referring to FIG. 4C, it illustrates scanning electron microscopy imagesin accordance with Experimental examples 1 and 4 and Comparative example1 of the present disclosure. As shown in FIG. 4C, Comparative example 1manifested a rough surface due to irregular albumin particles,Experimental example 1 exhibited smoother surface due to connection tothe COL, while Experimental example 4 was comparably the most smoothestdue to connection to the Mx.

Experimental Examples 1′and 4′ and Comparative Example 1′, 3, 4 and 5

Alterations in Post-Destruction Absorbance Spectra Before and After theModification and Drug Carriage of Microbubbles

Microbubbles were ultrasound contrast agents, which could give rise tothe effect of inertial cavitation and be destroyed under ultrasound (US)sonication of certain intensity and period of time. This study compared3 kinds of destroyed microbubbles, namely the destroyed MB (Comparativeexample 1′), destroyed COL-MB (Experimental example 1′), and destroyedMx-COL-MB (Experimental example 4′), to non-microbubble comparativeexamples, namely the albumin (Comparative example 3), the Mx(Comparative example 4), and the saline (Comparative example 5), to lookinto the alterations in post-destruction absorbance spectra before andafter the modification and drug carriage of microbubbles.

Referring to FIG. 5, it illustrates a diagram of absorbance spectra inaccordance with Experimental examples 1′ and 4′ and Comparative examples1′, 3, 4 and 5 of the present disclosure. As shown in FIG. 5, comparedto Comparative examples 3 and 4 which had the specific absorption peaks,neither Comparative example 1′ nor Experimental example 1′ absorbedlights of specific wavelengths, which was the same as Comparativeexample 5. However, Experimental example 4′ absorbed lights at thewavelength of 230 nm, 261 nm, and 285 nm, which was the same as theabsorption spectrum of Comparative example 4, indicating that thedestroyed Mx-COL-MB released Mx.

Experimental Examples 7-10

In vitro drug release study of the Mx-COL-MB

The study measured the Mx release pattern of the Mx-COL-MB throughdialysis method after ultrasound (US) treatment. The Mx-COL-MBs weredivided into 4 kinds of environments, which were the Experimentalexample 7 at pH 4 with ultrasound treatment, Experimental example 8 atpH 4 without ultrasound treatment, Experimental example 9 at pH 7.4 withultrasound treatment, and Experimental example 10 at pH 7.4 withoutultrasound treatment. The procedures of the study included: loading 3 mlof PBS suspension solution containing any one of the Experimentalexamples 7-10 into a dialysis bag, and dialyzing the suspension solutionagainst the PBS release medium at the same pH. The dialysis temperaturewas 37±0.5° C., accompanied with magnetic stirring at 600 rpm. At the0.5^(th) hr, the 1-MHz ultrasound sonicator was positioned 3 mm from thetop of the dialysis bag and performed ultrasound sonication at a powerdensity of 3 W/cm² for 1 min. At the time intervals of 0.1, 0.2, 0.3,0.4, 0.5, 1, 2, 3, 4, 5, and 6 hours, 1 ml of sample was taken out fromthe release medium to measure the content of Mx dialyzed into thesample, and the same volume of PBS was supplemented into the releasemedium. The accumulative release percentage of Mx was calculatedaccording to the following equation:

$R = {\frac{{c_{n}v_{0}} + {\sum_{i = 0}^{n - 1}{c_{i}v_{i}}}}{W} \times 100\%}$

where R is the release rate, c_(n) is the drug concentration in thetotal release medium at each time interval of sampling, c_(i) is thedrug concentration in the previous release medium sample, v₀ is thetotal volume of the release medium, v_(i) is the volume of the releasemedium sample, and W is the total drug content of the release sample.

Referring to FIG. 6, it illustrates a diagram of cumulative releaserates of Minoxidil against time in accordance with Experimental examples7-10 of the present disclosure. As shown in FIG. 6, the cumulative Mxrelease rate of Experimental example 10 at the 0.5^(th) hr was only13.6%, while the cumulative Mx release rate of Experimental example 9 atthe same time was 29.2%. The cumulative Mx release rate of Experimentalexample 8 at the 0.5^(th) hr was 30.3%, while the cumulative Mx releaserate of Experimental example 7 at the same time was 51.4%. Without USsonication, the Mx release was significantly limited. The cumulative Mxrelease rate of Experimental example 10 at the 6^(th) hr was only 41.2%,while the cumulative Mx release rate of Experimental example 8 at the6^(th) hr was 67.3%. However, after US treatment, the Mx-COL-MBconstantly and slowly released the Mx. The cumulative Mx release rate ofExperimental example 9 at the 6^(th) hr reached 68%, while thecumulative Mx release rate of Experimental example 7 at the 6^(th) hrreached 89%. Hence, US treatment elevated the Mx release rate of theMx-COL-MB by 20-26%, while pH 4 also elevated the Mx release rate. Sincethe pH of the human scalp is coincidently about pH 4.5-5.5, whenMx-COL-MB contacts the scalp with combined ultrasound sonication at 3W/cm² for 1 min, Mx can be rapidly released.

Experimental Example 11 and Comparative Examples 6-7

Study of Transdermal Penetration Depth

The study includes 3 groups of solutions: Comparative example 6 withoutMBs added and without US treatment, the US group without MBs added whilewith US treatment (Comparative example 7), and the US+MBs group with 500μl MBs added and US treatment (Experimental example 11). Each groupcontained 0.1 mg FITC (fluorescein isothiocyanate), and was uniformlyloaded onto the porcine ear skin with an area of 4.5 cm² and a thicknessof 3 mm, sonicated by the 1-MHz ultrasound sonicator successively atpower density of 3 W/cm², with each sonication period of 1 min. Aftersonication, the FITC (and the MBs) retained on the porcine ear skin for6 hr, and was then washed away.

Referring to FIG. 7, it illustrates scanning electron microscopy imagesof transdermal delivery of fluorescent agents in accordance withExperimental example 11 and Comparative examples 6 and 7 of the presentdisclosure. FIG. 7A-7C are bright-field images, and FIG. 7D-7F aredark-field fluorescent images. FIGS. 7A and 7D represent Comparativeexample 6, whose penetration depth of FITC was 312±19 μm. FIGS. 7B and7E represent Comparative example 7, whose penetration depth of FITC was405±23 μm. In contrast, FIGS. 7C and 7F represent Experimental example11, whose penetration depth of FITC reached 1856±45 μm. This indicatesthat after US treatment, the skin permeability is increased, and furtherwith the effect of the MB, transdermal penetration of drugs can beeffective.

Experimental Examples 4 and 12 And Comparative Examples 1, 4, 8 and 9

Transdermal Penetration Study of Mx Released by Mx-COL-MBs

The study included 6 groups of solutions: groups without ultrasoundtreatment including the Mx group (Comparative example 4), the MBs groups(Comparative example 1), and the Mx-COL-MBs group (Experimental example4), and groups with ultrasound treatment including the US+Mx group(Comparative example 8), the US+MBs+Mx groups (Comparative example 9),and the US+Mx-COL-MBs group (Experimental example 12). In Comparativeexamples 4, 8, and 9, the concentration of Mx was 0.3 mg/ml, while inExperimental examples 4 and 12, the concentration of Mx carried by theMx-COL-MB was 0.3 mg/ml. The procedures of the study included:interposing a piece of 3 mm-thick porcine ear skin between a donor celland a receptor cell of the Franz diffusion cell at a temperature of37±0.5° C. 1 ml of any one of the abovementioned 6 solutions was appliedto the donor cell facing the stratum corneum (SC) side, enabling the MB,Mx, or Mx-COL-MB to penetrate the Parafilm on a bottom of the donor celland reach the skin. Besides, the receptor cell facing the dermis sidewas filled with 12 ml of PBS at pH 7.4, accompanied with magneticstirring at the rate of 600 rpm and 0.01% gentamicin to preventbacterial degradation of the Mx during the penetration process. At the0.5^(th) hr, a 1-MHz ultrasound sonicator was positioned 3 mm from thetop of the skin, and the skin was sonicated at a power density of 3W/cm² for 1 min. At the time intervals of 0.5, 1, 2, 3, 4, 5, 6, 8, 12and 18 hours, 1 ml of receptor solution sample filtered by 0.2-μm poreswas extracted from the receptor cell to measure the Mx content in thereceptor solution, with the receptor solution refilled each time withthe same volume of PBS. After 18 hours, the porcine skin sample waswashed and then homogenized with 1 ml of the receptor solution. Thesupernatant was centrifuged to acquire further supernatant to measurethe absorbed amount of Mx in the porcine ear skin.

Referring to FIG. 8, it illustrates a diagram of cumulative penetrationamounts of Minoxidil against time in accordance with Experimentalexamples 4 and 12 and Comparative examples 1, 4, 8 and 9 of the presentdisclosure. As shown in FIG. 8, although Comparative example 9 inducedrapid Mx penetration with the amount of 62.9±5.0 μg/cm³ in the first 6hours, the penetration amount gradually sloped gently in 8-18 hours.Relatively, the Mx penetration amount of Experimental example 12 reached240.0±32.8 μg/ml at the 18^(th) hour, which was significantly higherthan the amount of 174.3±19.8 μg/ml in Comparative example 9, the amountof 113.0±6.0 μg/ml in Comparative example 8, the amount of 103.1±0.5μg/ml in Comparative example 4, the amount of 73.3±1.4 μg/ml inExperimental example 4, and the amount of 21.7±1.6 μg/ml (thebackground) in Comparative example 1. This indicated that afterultrasound treatment of the Mx-COL-MBs and MBs+Mx (i.e. Experimentalexamples 12 and 9), the Mx penetration amount could increase by 2.3 and1.7 folds compared to the Mx group (Comparative example 4) respectively.Therefore, in Experimental example 12, the US-treated Mx-COL-MB couldexhibit the highest amount of Mx penetration in 18 hours.

TABLE 2 Amount of Amount of Amount of skin skin total Skin weightabsorption penetration penetration Group (g) (μg/ml) (μg/ml) (μg/ml)Comparative 0.1659 ± 0.0325 52.42 ± 9.55 103.1 ± 0.51 155.54 ± 10.05example 4 Comparative 0.1557 ± 0.0198 33.02 ± 1.56 113.01 ± 5.95  126.04± 7.52  example 8 Comparative 0.1654 ± 0.0214 27.33 ± 2.73 174.34 ±14.01 201.68 ± 16.74 example 9 Experimental 0.1845 ± 0.0376 26.75 ± 3.9873.26 ± 1.42 100.01 ± 5.40  example 4 Experimental 0.1430 ± 0.0253 17.43± 1.12 240.04 ± 32.82 257.48 ± 33.94 example 12

Referring to the above Table 2, in terms of the amount of skinabsorption, the Mx amount of skin absorption reached 52.42±9.55 μg/ml inComparative example 4, which was significantly higher than Comparativeexample 8, Comparative example 9, Experimental example 4, andExperimental example 12. Nevertheless, in terms of the amount of skinpenetration, the amount of skin penetration in the Experimental example12 reached 240.04±32.82 μg/ml, significantly higher than the othergroups. Thus, Mx-COL-MBs with US treatments exhibits not only thehighest Mx amount of cumulative penetration, but also highest Mx amountof skin penetration, facilitating the amount of transdermally penetratedMx to be the highest.

Experimental Example 12 and Comparative Examples 4, 8, 9 and 10

Study of Animal Hair Growth

The study utilized 6-week-old C57BL/6 mice weighing 20-25 g, which wereshaved at an area of about 10 cm² in the back at 8 weeks old, when allof the hair follicles were synchronized in the telogen phase. The micewere divided into 5 groups including: the untreated group (Comparativeexample 10), the Mx group with only Mx provided (Comparative example 4),the US group with Mx and US treatment provided (Comparative example 8),the US+MBs group with Mx, MBs, and US treatment provided (Comparativeexample 9), the US+Mx-COL-MBs group with Mx-COL-MBs and US treatmentprovided (Experimental example 12). Among all groups, the US treatmentwas applied at 3 W/cm² for 1 min, and the concentration of Mx was 0.3mg/ml (i.e. 0.5 ml/cm²) in all groups. The change in skin luminosity ofthe shaved area was assessed using the Chroma Meter. The hair growthrate was calculated according to the following equation:

Hair growth rate (%)=(L ₁ −L _(n))/L ₁×100%

where L₁ is the luminosity index of the shaved area, and L_(n) is theluminosity index of the area at different time intervals.

Referring to FIG. 9, it illustrates top-view observation images of hairgrowth of mice backs against time in accordance with Experimentalexample 12 and Comparative examples 4, 8, 9 and 10 of the presentdisclosure. As show in FIG. 9, at day 10, the numbers of mice withsignificantly lower skin luminosity were: 5 in Experimental example 12,3 in Comparative example 9, 1 in Comparative example 8, 1 in Comparativeexample 4, and 0 in Comparative example 10, indicating the most rapidhair growth in Experimental example 12. At day 14, hair growth rates ofeach mouse in Experimental example 12 were more significant than therates of the rest four groups.

Referring to FIG. 10, it illustrates a diagram of hair growth rates ofmice backs against time in accordance with Experimental example 12 andComparative examples 4, 8, 9 and 10 of the present disclosure. As shownin FIG. 10, at day 10 and 14, the hair growth rate of Experimentalexample 12 increased by 22.6% and 64.7% respectively compared to day 1,significantly higher than the rates of the rest four groups. At day 16,the hair growth rate of Experimental example 12 reached 84.2%, higherthan the 49.2% of Comparative example 10, the 45.5% of Comparativeexample 4, the 64.5% of Comparative example 8, and the 83.5% ofComparative example 9.

Referring to FIG. 11, it illustrates scanning electron microscopyhistological images of mice backs in accordance with Experimentalexample 12 and Comparative examples 4, 8, 9 and 10 of the presentdisclosure. FIG. 11A-11E demonstrate vertical view of hair follicles,and FIG. 11F-11J demonstrate coronal view of hair follicles. FIGS. 11Aand 11F illustrate Comparative example 10. FIGS. 11B and 11G illustrateComparative example 4. FIGS. 11C and 11H illustrate Comparative example8. FIGS. 11D and 11I illustrate Comparative example 9. FIGS. 11E and 11Jillustrate Experimental example 12. As shown in FIG. 11A-11E, at day 21,Comparative example 4 exhibited significantly increased skin thickness,Comparative example 8 exhibited increased hair length, Comparativeexample 9 exhibited hair growth with even longer hair, whileExperimental example 12 exhibited the most significantly increased skinthickness and hair length with promoted elongation of hair folliclesfrom the epidermis down to the subcutis in a vertical section. In FIG.11F-11J, although the number of hair follicles didn't increasesignificantly after treatments among all groups, Comparative example 8,Comparative example 9, and Experimental example 12 all exhibitedincrease in the diameter of the keratinized hair shafts and the size ofhair follicles, with Comparative example 9 and Experimental example 12showing a more significant level of increase, and Experimental example12 showing the most significant increase in the elongation of the hairfollicle section.

In summary, the present disclosure applies the drug-carrying modifiedmicrobubble (Mx-COL-MB) dissolved in PBS to skin externally, andperforms ultrasound (US) treatment to prompt the Mx-COL-MB to release agreat amount of Minoxidil (Mx), while the Mx can undergo transdermalpenetration to reach the hair follicle to significantly enhance the hairgrowth rate, which achieves the effects of shortening the course of hairgrowth treatments and reduction in the sensitive skin reactions.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the structure of the presentdisclosure without departing from the scope or spirit of the disclosure.In view of the foregoing, it is intended that the present disclosurecover modifications and variations of this disclosure provided they fallwithin the scope of the following claims.

What is claimed is:
 1. A modified microbubble, comprising: an albuminmicrobubble, comprising: an albumin shell; and a gas core inside thealbumin shell; and a plurality of chitosan oligosaccharide lactatesconnected to an outer surface of the albumin shell.
 2. The modifiedmicrobubble of claim 1, further comprising a drug connected to thechitosan oligosaccharide lactate to form a drug-carrying modifiedmicrobubble.
 3. The modified microbubble of claim 2, wherein the drughas a negative charge.
 4. The modified microbubble of claim 2, whereinthe drug is Minoxidil.
 5. The modified microbubble of claim 1, themodified microbubble has a diameter of 4,000 nm to 4,400 nm.